There is increasing evidence that the intervening sequences (IVS) of eukaryotic genes play a significant role in eukaryotic gene expression. The nucleotide sequences at the IVS boundaries are highly conserved in evolution, and mutations at these boundaries are associated with loss of specific gene function. I propose to study the role of IVS, both at their boundaries and within these genes in the expression of the human Beta globin genes. This system has several advantages including: 1) Availability of clones of DNA containing genomic Beta globin genes and their IVS; 2) Knowledge of the entire nucleotide sequence of the Beta globin gene; 3) Tissue culture systems for expression of these genes; and 4) The availability of mutant Beta globin genes which appear to be defective in IVS processing. I intend to use a monkey kidney tissue culture system to analyze transcripts produced by a compound plasmid containing SV40 and pBR sequences, and either normal or abnormal cloned human Beta globin gene fragments. The RNA transcripts will be analyzed by S1 mapping of the spliced and unspliced products of transcription using a variety of DNA cloned probes. The effects of mutations in the IVS on the structure of RNA transcripts will be studied using both site directed mutations in normal genes, as well as an available Beta+ thalassemia gene recently isolated in this laboratory which appears to be defective in processing. A defect in a Beta+ thalassemia gene involving the small intervening sequence (IVS 1) of the Beta globin gene has recently been reported. Preliminary data on another clone of DNA from a patient with Beta+ thalassemia that I have been studying indicates that a defect in the large intervening sequence (IVS 2) is associated with an abnormal RNA transcript, suggesting a splicing or processing mutation. My proposed research, therefore, is to define the normal process of splicing in the human Beta globin gene, and to use the Beta+ thalassemia gene to determine the defect in processing resulting from changes in nucleotide sequence of this gene. In the long term, the proposed research should expand our knowledge of the way in which changes in DNA sequence affect the expression of eukaryotic genes and lead to inherited disorders. Specifically, it may contribute to our understanding of Beta+ thalassemia at the molecular level, and may provide insights which will lead to new approaches to the treatment of these disorders.